Radical Pair States

4 Radical Pair States. - So far we have discussed single radical-ions that occur in the charge-separation process and can be trapped under certain condition in bRC or can even be chemically generated. The initial light-induced charge separation creates radical pairs (RPs) that can be studied by time-resolved transient or pulse EPR  

The first observable RP P + 1 a is short-lived ( 200 ps) and can only be observed by EPR in mutant RCs with an increased lifetime. Therefore, most of the work has been performed on the secondary RP, namely P + V, which has a lifetime in the ps range. Due to the very slow ET from Q to QB the tertiary RP is not observed. However, under certain conditions a state 4 b can be 

In the spin-correlated RP the two radicals interact via electron-electron dipolar and exchange interaction which leads to line splitting. The ET process creates the RP in a strongly spin-polarized state with a characteristic intensity pattern of the lines that occur either in enhanced absorption (A) or emission (E).144 145 The spectrum is therefore very intense and can directly be observed with cw EPR (transient EPR) or by pulse methods (field-swept ESE).14 To study the RPs high field EPR with its increased Zeeman resolution proved to be very useful the first experiment on an RP was performed by Prisner et al. in 1995146. From the analysis of the RP structure detailed information about the relative orientation of the two radicals can be extracted from the interaction parameters. In addition kinetic information about the formation and decay of the RP and the polarization are available (see references 145,147). 

The application of time-resolved pulse ENDOR to study the spin-polarized short-lived RP has been an important advancement. The feasibility of such 

In the case of the carotenoid-containing LH2 complex, the triplet states of BChl a and carotenoid (spheroidene) were generated immediately after excitation, but the triplet-state BChl a was quenched efficiently by the carotenoid so that no BChl a cation-radical was generated. Thus, the photoprotective function of the carotenoid in this antenna complex has been proven. 

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It has been suggested that P BChl (where BChl is one of the two monomeric or "accessory BChls that are not part of P) is a transient state prior to P "I (14,16,19), although the evidence supporting this view has been criticized (23, 24) Recent subpicosecond studies find no evidence for P "BChl (8,9) These new results do not preclude some involvement of a monomeric BChl in the early photochemistry, only that P BChl apparently is not a kinetically resolved transient state Perhaps P itself contains some charge-transfer character between its component BChls, or between P and one or both of the monomeric BChls (8,9,25-27) One of the two monomeric BChls apparently can be removed by treatment of the reaction center with sodium borohydride (28) and subsequent chromatography, with no impairment of the primary electron transfer reactions (29) Thus, at present it appears that P I is the first resolved radical-pair state, and it forms with a time constant of about 4 ps in Rps sphaeroides ... 

The participation of such ionic states would load one to expect disproportionation rates to increase in the series CH8, C2H5J iso-C8H7, and ferf-butyl, since the ionization potentials of these radicals form a descending series 10.0 e.v., 8.8 e.v., 7.9 e.v., and 6.9 e.v., respectively. This effect of decreasing ionization potential, which might be expected to push the ion-pair state down in energy, considerably below the radical pair state, is considerably off-set by the increasing value of rt (eqs. 5 and 6). ... 

With each type of PS-II membrane we have measured the absorption increase at 820nm induced by a 20-ps laser flash, first under oxidizing conditions the signal then is due to P-680+ in all the reaction centers. The measurement was then effected under the same conditions, except that QA was reduced the signal then is due to (P-680+, I-) or to excited chlorophyll. The results are as follows (n is the number of chlorophylls per P-680 or antenna size is the half-time of the signal obtained when Q, is reduced b is the ratio (P -680+, r/P-680+ under reducing conditions, i.e. the fraction of reaction centers which are in the radical pair state 1-b is thus the fraction of reaction centers where chlorophyll is excited alternatively, a is the same ratio if we suppose that there is no excited chlorophyll detected in our experiments) ... 

At room temperature, the P state decays by electron transfer from P to the Ha BPhe, located approximately half-way across the membrane, forming the radical pair state P Ha . Why electron transfer in the reaction centre proceeds exclusively along the A-branch of cofactors, despite the approximately symmetrical arrangement of the cofactors in the A- and B-branches, is a matter of ongoing debate (see Section 5.2), as is the role... 

Rb. sphaeroides reaction centre, the electron is provided by a cytochrome C2 that docks to the periplasmic face of the reaction centre. In the absence of this reduction of P, the radical pair state recombines to the ground... 

The initial electron acceptor can be made to accumulate in the reduced state (I ) if reaction centers which have bound (or added) cytochromes are illuminated continuously after the reduction of [56,67-69]. Each time the radical-pair state P I is formed, has a brief opportunity to oxidize the cytochrome instead of recovering an electron from I. The probability of electron transfer from the cytochrome is low, because the back reactions between P and I are much faster than the cytochrome oxidation. After many turnovers, however, essentially all of the reaction centers may be left with I reduced, particularly if the return of electrons... 

The radical-pair state P I is also formed if unreduced reaction centers are excited with a short flash, but it then decays with a time constant of about 200 ps [54,70-74]. The rapid decay of the transient state presumably reflects electron transfer from I to (Fig. 1), because it is prevented if the quinone is already reduced or is extracted from the reaction centers. The transient absorption changes suggest that I is a BPh 7r-radical anion, which interacts with a nearby BChl [75-77], The absorbance changes in a band associated with the BChl decay with somewhat different kinetics from those in bands associated with the BPh or BPh , perhaps because they reflect nuclear motions in the electron carriers or the surrounding protein [75]. The possible role of the BChl in the initial transfer of an electron from P to the BPh will be discussed below. 

In radical-pair states, the average distance between the two unpaired electron spins is typically much larger than in triplet states. Hence, TREPR spectra of photogenerated (and electron-spin polarized) radical pairs are narrower due to the reduced mutual dipolar and exchange interactions as compared to flavin triplets. This is shown in Fig. 7b, where the TREPR signal of a flavin-based radical pair in photolyase is depicted [19]. Analysis of the spectral shapes of TREPR signals yields information on the chemical nature of the individual radicals of the radical-pair state, and the interaction of the radicals with each other and with their immediate surroundings. 

The TREPR results clearly show that cryptochromes (exemplified for the DASH-type) readily form radical-pair species upon photoexcitation. Spin correlation of such radical-pair states (singlet vs triplet), which is a necessary condition for magnetoselectivity of radical-pair reactions, manifests itself as electron-spin polarization of EPR transitions, which can be directly detected by TREPR in real time. Such observations support the conservation of photo-induced radical-pair reactions and their relevance among proteins of the photolyase/cryptochrome family. The results are of high relevance for studies of magnetosensors based on radical-pair (photo-)chemistry in general [114], and for the assessment of the suitability of cryptochrome radical pairs in animal magnetoreception in particular [17, 115]. 

The access to the mechanism of the primary reaction rests on two classes of spectroscopic approaches Direct experiments reflecting primary ET within its time domain and recombination experiments on the radical pair state (P+H ) which set restrictions for either mechanism. In this paper we summarize some recent results obtained along the line of both approaches and discuss their mechanistic implications. Unless stated otherwise, we refer to data from RCs of Rb.sphaeroides. 

For any description of the ET mechanism the energetics (i.e. the free energies and the reorganisation energies) of the states involved are crucial. Information on these can be derived from recombination experiments on the radical pair state P+H", see section 3.A. In addition, the recombination parameters allow some mechanistic insight since they set boundary conditions for the prevalence of either mechanism (1) and (2), see section 3.B. 

To identify and characterize this signal, MIA-spectra were measured (Figure 2). All resonances mentioned above cause absorption changes at the same wavelengths, thus the low frequency microwaves do influence a state within the electron transfer chain of the reaction center. We assume, that this state is the radical pair state P H and therefore we call the signal RP signal . 

To examine the properties of the radical pair state, we performed modified RYDMR experiments by applying low magnetic fields during the ADMR experiment. The intensity changes of the ADMR signals are shown in Figure 5. 

The most prominent feature of the temperature dependences depicted in Fig.2 is the superposition of two processes, one being thermally activated. The qualitative result of a detailed analysis [12] is the competition between the activationless direct ET of P Qa PQa and an activated recombination, e.g. via the radical pair state P Ha-... 

Figure la shows the spectrum of the flash induced absorption transient observed in the PS2 reaction centre preparation with a monoexponential lifetime of (1.0 0.1)ms under anaerobic conditions. This spectrum, although more detailed, is similar to that reported by Takakashi et al. at 50K [1]. The quantum yield of this triplet is approximately 30%. This transient is attributed to the P680 triplet state, formed by charge recombination from the primary radical pair state. This assignment is supported by the following data. 

ON A PRESUMED LONG-LIVED RELAXED RADICAL PAIR STATE IN CLOSED PHOTOSYSTEM H,... 

A net Coulombic repulsion between and the radical pair could result in an upward energy shift of the radical pair state compared to open reaction centres. This would result in a lower yield of radical pair formation (3), If Q. is double reduced and protonated ("jammed" reaction centre), the free energy of the radical pair state would resemble that in open centres. Consequently, the yield of radical pair formation and triplet would increase upon double reducing Q. ... 

On a Presumed Long-Lived Relaxed Radical Pair State in Qosed Photosystem II 443... 

Triplet states also can form by back reactions of radical-pair states that are created by photochemical electron transfer. We discuss this process briefly in Box 10.2. 

Booth, P.J., Crystall, B., Ahmad, I., Barber, J., Porter, G., et al. Observation of multiple radical pair states in photosystem 2 reaction centers. Biochemistry 30,7573-7586 (1991)... 

There seems to be general agreement now about D being of the order of 5 to 10 G. This corresponds well to the 24 MHz (8.6 G) which we find for this precursor state. Therefore we tentatively assign this newly observed resonance to the D + E transitions ( E being close to zero [13]) of the radical pair state. 

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